| The mechanism of burn wound healing is a complicated process which is quite differentfrom other types of wound such as incision, laceration or chronic ulcer. The patients willconfront lots of problems, but the key points they have to face are how to advance thehealing time and reduce the scar formation. So how to deal with the wound and shortenwound course are the major challenges the doctors should be facing. Healing, maintain thebody integrity by remodeling the damaged tissue, one is through the function of the bodyitself, the other is through the external intervention to accelerate the natural healing process.The external intervention containing topic drugs, dressings or surgery, and PDGF-BB iswidely believed to be the driving force behind wound healing but the high cost and risk ofcancer have limited its use in clinical settings.Besides, TGF-β3is widely expressed in tissuesand it could inhibit the effects of PDGF-BB on wound-healing. So the mechanism that drives skin cell migration into the wound bed when TGF-β3is abundantly expressed in tissues hasremained to be elusiveThe latest research showing that hsp90α plays an important role in wound healing. Hsp90α,which is induced by heat, alcohol, UV radiation, oxidants and other stresses, plays severalimportant roles in tissue repair. It is one of the most abundant molecular chaperones and canmaintain cell stability, assist the nascent proteins on correct folding, redirect misfiledproteins and cause the degradation of diverse set of client proteins. Comparing to the growthfactors, it can not only promote the epidermal cells migration, but also promote the dermalfibroblasts. It will also not be influenced by TGF-β3. In addition, hsp90α will also promotehealing in high glucose conditions.Despite the proposed role for hsp90αin wound healing,details of its mechanism are lacking, and there has been no systematic exploration of therelationship between burn injury and hsp90α. Whether it can accelerate the burn woundhealing by promoting cell proliferation and migration worth to be discussion.This research shows the importance of hsp90α in accelerating cell migration,proliferationand wound healing following burn injury. Differences in hsp90α expression were observedaround the edge of the wound over time. Hsp90α was shown to accelerate heat shock skincell migration and proliferation in HaCaT cells. Highly purified recombinant hsp90αwasapplied to deep second-degree burn injury, and mice showed accelerated wound healing atevery point in time, with no adverse effectsobserved. Consequently, hsp90α is a promisingcandidate for burn wound healing therapy.1. Purpose:To systematic exploration the relationship between burn injury and hsp90α, and to find thatwhether it can accelerate the burn wound healing by promoting cell proliferation andmigration.2. Methods:2.1.85male BALB/c mice (20±1g) were randomly divided into5groups. The controlgroup (n=5), steam scaded for2,4,6and8s groups(n=20).Then they were anesthetized with1%pentobarbital sodium using peritoneal injection followed by unhairing with10%sodium sulfide24h before scalding. Hot steam and home-made nozzlewere employed forscalding on the back of the mice for the indicating timethrough a2cm hole. Skin sampleswere obtained immediately or12,24and48h after scalding and sequentially subjected tohematoxylin and eosin stain to study the relationship between burn depth and scalding time.2.2.25male BALB/c mice (20±1g) were randomly divided into2groups.Immunohistochemisty group (n=5) and Transcutaneous Oxygen Tension measurementsgroup (n=20). They were performed to hypoxia examination on burn wound.Differencesbetween experimental and controlgroups were determined using one way ANOVA testing.P﹤0.05was considered statistically significant.2.3.55male BALB/c mice (20±1g) were randomly divided into3groups.Immunohistochemisty group (n=10) were sacrificed12h and48h after burn. Wounded andunwoundedskin tissues (1:1) were treated with PBS and hsp90α mAbs, The mice used forreal-time PCR group(n=24) were sacrificed0,0.5,1,3,6,12and24h after burns,full-thickness skin tissues were excised around the edges of each wounds and Western-blotgroup (n=21) were scarificed6,12,24,48,72and7d after burns.2.4. HaCaT cells wereplated in6-well tissue culture plates and then placed in a water bath at45°C for15minutes. Total cellular RNA was isolated at0,0.5,1,3,6,12and24h forReal-time PCR. HaCaTcells were divided into three groups:(1) control group,(2)8μg/mlhsp90α group and (3)0.5μmol/l17-DMAG group. Used for Scratch assays, cell cycledistributions and apoptosis analysis.2.5. Six-week-old male balb/c mice weighing20g each. Thirty animals were selected atrandom anddivided into three groups: a burn group treated with topical saline (Control, n=10), a burn group treated with topical hsp90α (Hsp90α, n=10), and a burn group treatedwith i. p. of17-DMAG before burning (17-DMAG, n=10). Images of wounds were taken at0,5,9,13and21days after burning. On day7, half of the animals were sacrificed andfull-thickness skin and underlying adherent tissue samples were excised and fixed informalinovernight and subjected to HE staining. 3. Results:3.1. The burn depth is closely related to scalding time, after scalding for4s, a superfcial anddeep partial thickness injury was observed. The depth was observed to deepen with time andbe stable at24h after scalding. This result indicates that histological analysis of burn depthshould be performed at least24h later after scalding.3.2. Hypoxia was found in burn wounds and especially in the margin of the wounds. Theoxygen partial pressure of normal mice was about53.0±2.4mmHg and decreased to about37.1±2.7mmHg in deep-second degree burned mice. Immunohistochemisty resultsrevealed hypoxia was found in the marginal zones of the wounds.3.3. To assess hsp90α gene expression during early burn wound, skin tissues were harvestedat the indicating time after burn. Real-time PCR analysis revealed that hsp90α expressionlevels peaked at6h and were20times higher than those of control mice.Hsp90α expressionwas also assessed at the protein level. It was followed by agradual increase up until72hafter the infliction of the burn.3.4. Total cellular RNA isolated from heat shock cells conformed the same results that it waspeaking at0.5h after heat shock.Scratch assays found that the hsp90α group showed anaccelerated decrease in gap size, almost complete gap closure at24h. Conversely, the17-DMAG group showed only slight closure of the scratch (p <0.05). Heat shock of cellsthat were pre-exposed to hsp90α exhibited an obviously decrease in the number of cells inthe G1, whereae17-DMAG induced G1cell cycle arrest. Furthermore, the rate of apoptosisof HaCaT, was significantly reduced for cells treated with hsp90α, whereas17-DMAGelicited the opposite effect (p <0.05).These results provide clear confirmation of theimportance of hsp90α in heat shock cell viability.3.5. To examine the therapeutic potential of hsp90α, we evaluated the effects of hsp90α onburn wound healingby histological and macroscopic observation. For the hsp90α group, thewound healing was more rapid, with progressively smaller wounds on all observation days(P＜0.05). Mice treated with17-DMAG had a healing response that was slower than thecontrol group. It can be concluded that hsp90α accelerates wound closure significantly andshortens the time required for healing. 4. Conclusions:4.1. A stable deep-second degree burn modelcould be established with20g mice by scaldingfor4s which was confirmed by pathology results of the burned skin samples that wereobtained at24h after burn.4.2. Hypoxia was found in burn wounds and especially in the marginal zones of the wounds.4.3. Burn injury stimulates the edges of the wound area to secrete hsp90α transiently at boththe RNA and protein level.4.4. Extracellular hsp90α showed an positive effect onskin cell migration and proliferation inan in vitro heat shock model, the effect could be inhibited by17-DMAG.4.5. After wound treated by hsp90α, inflammation was reduced, granulation tissue showedsignificant development, and the epidermal cells at the wound margins progressed morerapidly than in other groups. Hsp90α can promote burn wound healing by acceleratingre-epithelialization and granulesformation. |
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